Cytokine polypeptides and antibodies containing a signal sequence for the attachement of glycosylphosphatidylinositol

ABSTRACT

The invention relates to chimeric recombinant polypeptides, preferably therapeutic polypeptides, for example cytokines or antibodies, which are engineered to include a signal sequence for the attachment of glycosylphosphatidylinositol; cells expressing said polypeptides and methods to manufacture said polypeptides.

The invention relates to chimeric recombinant polypeptides, preferablytherapeutic polypeptides, which are engineered to include a signalsequence for the attachment of glycosylphosphatidylinositol; cellsexpressing said polypeptides and methods to manufacture saidpolypeptides.

GPI-anchors are post-translational modifications to proteins that addglycosylphosphatidylinositol which enable these proteins to anchor tothe extracellular side of cell membranes. Typically, extracellularproteins which have a GPI anchor do not have transmembrane orcytoplasmic domains. GPI anchor proteins occur in all eukaryotes andform a diverse variety of proteins that includes by example and not byway of limitation, membrane associated enzymes, adhesion molecules andproteins which coat the outer surface of protozoan parasites such asTrypanosoma brucei spp. The human kidney includes a number of examplesof GPI-anchored proteins i.e. uromodulin, carbonic anhydrase type IV,alkaline phosphatase, Thy-1, BP-3, amino peptidase P, anddipeptidylpeptidase.

All GPI-anchor proteins are initially synthesized with a transmembraneanchor which, after translocation across the endoplasmic reticulum, iscleaved and covalently linked to a preformed GPI anchor by a specifictransamidase enzyme. The modification of proteins by the addition of aGPI-anchor confers important properties on the protein since theaddition of the lipid moiety allows the protein to be inserted into cellmembranes thereby anchoring the protein thus increasing its effectivelocal concentration.

There are some general requirements for creating a synthetic GPI anchorsequence. These are a hydrophobic region at the C-terminus of themolecule (10-20 amino acids) not followed by a cluster of basicresidues, a “spacer domain” of 7-10 residues preceding the hydrophobicregion and small amino acids after the spacer region, where cleavage ofthe precursor and attachment of the anchor occurs. The GPI anchor ispreassembled and added to nascent protein in the endoplasmic reticulum.

Concomitant with this step, the initial C-terminal peptide is removed sothat the GPI anchor is covalently attached to a new C-terminal aminoacid on the protein.

The large scale production of recombinant proteins requires a highstandard of quality control since many of these proteins are used aspharmaceuticals, for example: growth hormone; leptin; erythropoietin;prolactin; TNF, interleukins (IL), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-9, IL-10, IL-11; the p35 subunit of IL-12, IL-13, IL-15; granulocytecolony stimulating factor (G-CSF); granulocyte macrophage colonystimulating factor (GM-CSF); ciliary neurotrophic factor (CNTF);cardiotrophin-1 (CT-1); leukemia inhibitory factor (LIF); oncostatin M(OSM); interferon, IFNα, IFNγ, and extracellular receptor domains fromany cell surface receptor. Moreover, the development of vaccines,particularly subunit vaccines, (vaccines based on a defined antigen, forexample gp120 of HIV), requires the production of large amounts of pureprotein free from contaminating antigens which may provoke anaphylaxis.

The production of recombinant protein in cell expression systems isbased either on prokaryotic cell expression or eukaryotic cellexpression. The latter is preferred when post-translation modificationsto the protein are required. Eukaryotic systems include the use ofmammalian cells, e.g. Chinese Hamster Ovary cells; insect cells e.g.Spodoptera spp; or yeast e.g. Saccharomyces spp, Pichia spp.

We disclose recombinant proteins which are adapted by the addition of asignal sequence for the attachment of glycosylphosphatidylinositol.Transfected cells expressing said adapted protein retain biologicalactivity and advantageously can be purified with relative ease due totheir location in the cell membrane (and/or shedding into the culturemedia facilitating continuous culture methods) of cells transfected withnucleic acid molecules, typically vectors, which express these proteins.

According to an aspect of the invention there is provided a chimericpolypeptide wherein said polypeptide is engineered to include a domaincomprising at least one heterologous signal sequence which sequencedirects the attachment of at least one glycosylphosphatidylinositolmolecule.

In our co-pending application, PCT/GB02/04665, currently unpublished, wedisclose antagonistic chimeric polypeptides which comprise the fusion ofa ligand binding domain of a cytokine receptor and a domain whichincludes a signal sequence for the attachment ofglycosylphosphatidylinositol. The content of PCT/GB02/04665 is herebydisclaimed with respect to the present application.

In a further preferred embodiment of the invention said domain comprisesthe amino acid sequence: PSPTPTETAT PSPTPKPTST PEETEAPSSA TTLISPLSLIVIFISFVLLI.

In an alternative preferred embodiment of the invention said domaincomprises the amino acid sequence: LVPRGSLEGR GTSITAYNSE GESAEFFFLLILLLLLVLV.

In a further alternative preferred embodiment of the invention saiddomain comprises the amino acid sequence: TSITAYKSE GESAEFFFLLILLLLLVLV.

In a preferred embodiment of the invention said polypeptide includes atleast one glycosylphosphatidylinositol molecule.

In a further preferred embodiment of the invention said polypeptide is atherapeutic polypeptide.

Typically a therapeutic polypeptide is a polypeptide with agonistic orantagonistic activity.

For example, and not by way of limitation, tumor suppressor polypeptides(e.g. p53 polypeptide, the APC polypeptide, the DPC-4 polypeptide, theBRCA-1 polypeptide, the BRCA-2 polypeptide, the WT-1 polypeptide, theretinoblastoma polypeptide (Lee, et al. (1987) Nature 329:642), theMMAC-1 polypeptide, the adenomatous polyposis coli protein (U.S. Pat.No. 5,783,666), the deleted in colon carcinoma (DCC) polypeptide, theMMSC-2 polypeptide, the NF-1 polypeptide, nasopharyngeal carcinomatumour suppressor polypeptide (Cheng, et al. 1998. Proc. Nat. Acad. Sci.95:3042-3047), the MTS1 polypeptide, the CDK4 polypeptide, the NF-1polypeptide, the NF2 polypeptide, and the VHL polypeptide.

“Antigenic polypeptides” (e.g. tumour rejection antigens the MAGE, BAGE,GAGE and DAGE families of tumour rejection antigens, see Schulz et alProc Natl Acad Sci USA, 1991, 88, pp 991-993). Antigenic polypeptidesalso includes polypeptide antigens used in the preparation of vaccineswhich provide protection against infectious agents. For example, virusessuch as Human Immunodeficiency Virus (HIV1 & 2); Human T Cell LeukaemiaVirus (HTLV 1 & 2); Ebola virus; Human Papilloma Virus (e.g. HPV-2,HPV-5, HPV-8 HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54 and HPV-56);papovavirus; rhinovirus; poliovirus; herpesvirus; adenovirus; EpsteinBarr virus; influenza virus, hepatitis B and C viruses. Antigens derivedfrom pathogenic bacteria such as Staphylococcus aureus; Staphylococcusepidermidis; Enterococcus faecalis; Mycobacterium tuberculsis;Streptococcus group B; Streptoccocus pneumoniae; Helicobacter pylori;Neisseria gonorrhea; Streptococcus group A; Borrelia burgdorferi;Coccidiodes immitis; Histoplasma sapsulatum; Neisseria meningitidis;Shigella flexneri; Escherichia coli; Haemophilus influenzae. Antigensderived from parasites such as Trypanosoma spp, Plasmodia spp,Schiztosoma spp; and pathogenic fungi such as Candida spp.

Therapeutic polypeptides which are “cytotoxic polypeptides” (e.g.pseudomonas exotoxin, ricin toxin, diptheria toxin).

Therapeutic polypeptides which are “cytostatic polypeptides” (e.g. p21,the retinoblastoma polypeptide, the E2F-Rb polypeptide, cyclin dependentkinase inhibitors such as P16, p15, p18 and p19, the growth arrestspecific homeobox (GAX) polypeptide as described in Branellec, et al,see WO97/16459 and WO96/30385.

Therapeutic polypeptides which are “pro-drug” activating polypeptides(e.g. cytosine deaminase).

Therapeutic polypeptides which are “apoptosis inducing” polypeptides(e.g. p53, adenovirus E3-11.6K(10.5K), the adenovirus E4orf4polypeptide, p53 pathway polypeptides, and caspases.

Therapeutic polypeptides which are “pharmaceutical polypeptides”(cytokines e.g. growth hormone; leptin; erythropoietin; prolactin; TNF,interleukins (IL), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10,IL-11; the p35 subunit of IL-12, IL-13, IL-15; granulocyte colonystimulating factor (G-CSF); granulocyte macrophage colony stimulatingfactor (GM-CSF); ciliary neurotrophic factor (CNTF); cardiotrophin-1(CT-1); leukemia inhibitory factor (LIF); oncostatin M (OSM);interferon, IFNα and IFNγ, and antagonists based on extracellular domainreceptor for the above cytokines or fusions of the above cytokines withtheir cognate extracellular domain receptor.

Therapeutic polypeptides which are “anti-angiogenic” polypeptides (e.g.angiostatin, inhibitors of vascular endothelial growth factor (VEGF)such as Tie 2 (as described in PNAS (USA)(1998) 95:8795-8800),endostatin.

Also included within the scope of therapeutic polypeptides aretherapeutic antibodies. Preferably said antibodies are monoclonalantibodies or at least the active binding fragments thereof. Therapeuticantibodies may be antibodies which bind and inhibit the activity ofbiological molecules, e.g. ligands or receptors.

Monoclonal antibodies may be humanised or chimeric antibodies. Thechimeric polypeptide therapeutic antibodies may be fusions withextracellular domain receptor polypeptides.

A chimeric antibody is produced by recombinant methods to contain thevariable region of an antibody with an invariant or constant region of ahuman antibody. A humanised antibody is produced by recombinant methodsto combine the complementarity determining regions (CDRs) of an antibodywith both the constant (C) regions and the framework regions from thevariable (V) regions of a human antibody.

In a preferred embodiment of the invention said fragment is a Fabfragment.

In a further preferred embodiment of the invention said antibody isselected from the group consisting of: F(ab′)₂, Fab, Fv and Fdfragments; and antibodies comprising CDR3 regions.

Preferably said fragments are single chain antibody variable regions(scFV's) or domain antibodies. If a hybridoma exists for a specificmonoclonal antibody it is well within the knowledge of the skilledperson to isolate scFv's from mRNA extracted from said hybridoma via RTPCR. Alternatively, phage display screening can be undertaken toidentify clones expressing scFv's. Domain antibodies are the smallestbinding part of an antibody (approximately 13 kDa). Examples of thistechnology is disclosed in U.S. Pat. No. 6,248,516, U.S. Pat. No.6,291,158, U.S. Pat. No. 6,127,197 and EP0368684 which are allincorporated by reference in their entirety.

A modified antibody, or variant antibody, and reference antibody, maydiffer in amino acid sequence by one or more substitutions, additions,deletions, truncations which may be present in any combination. Amongpreferred variants are those that vary from a reference polypeptide byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid by another amino acid of likecharacteristics. The following non-limiting list of amino acids areconsidered conservative replacements (similar): a) alanine, serine, andthreonine; b) glutamic acid and asparatic acid; c) asparagine andglutamine d) arginine and lysine; e) isoleucine, leucine, methionine andvaline and f) phenylalanine, tyrosine and tryptophan. Most highlypreferred are variants which show enhanced biological activity.

In a further preferred embodiment of the invention said therapeuticantibody, or active binding fragment thereof is fused, by a linkingmolecule, to said domain comprising a heterologous signal sequence whichsequence directs the attachment of at least oneglycosylphosphatidylinositol molecule. Preferably said linking moleculeis a peptide linking molecule.

Peptide linking molecules are known in the art, for example, repeats ofthe sequence Gly Gly Gly Gly Ser. For example, linking molecules whichcomprise, 1, 2, 3 or 4 repeats of said sequence. Linking molecules aredescribed in WO01/96565. Linking molecules may be flexible or rigid.

It is known that there are problems associated with the production ofbinding fragments of antibodies, in particular scFV's. These fragmentshave a tendency to aggregate in solution making their production on alarge scale problematic. The fusion of a domain comprising aglycosylphosphatidylinositol motif to a scFV's would facilitateproduction and purification.

In a further preferred embodiment of the invention there is provided apolypeptide according to the invention which has been modified byaddition, deletion or substitution of at least one amino acid residue toprovide a sequence variant of the polypeptide according to theinvention.

Typically, variants include chimeras specifically modified to alter afeature of the polypeptide unrelated to its physiological activity. Forexample, cysteine residues can be substituted or deleted to preventunwanted disulfide linkages. Similarly, certain amino acids can bechanged to enhance expression of the chimera by eliminating proteolysisby proteases in an expression system.

The skilled person will also realize that conservative amino acidsubstitutions may be made in the chimeric polypeptides to providefunctionally equivalent variants of the foregoing polypeptides, (i.e.the variants retain the functional capabilities of the chimeras). Asused herein, a “conservative amino acid substitution” refers to an aminoacid substitution which does not deleteriously alter the relativecharge, hydrophobicity or size characteristics of the protein in whichthe amino acid substitution is made.

Variants can be prepared according to methods for altering polypeptidesequence known to one of ordinary skill in the art such as are found inreferences which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D. (Q,N can be included. in someinstances where size and polarity are conserved, but chelation isunimportant)

Conservative amino-acid substitutions in the amino acid sequence ofchimeric polypeptides to produce functionally equivalent variants ofthese polypeptides typically are made by alteration of a nucleic acidencoding the chimera. Such substitutions can be made by a variety ofmethods known to one of ordinary skill in the art. For example, aminoacid substitutions may be made by PCR-directed mutation, site-directedmutagenesis according to the method of Kunkel, Proc. Nat. Acad. Sci.U.S.A. 82: 488-492, 1985.

Alternatively, or preferably, said modification includes the use ofmodified amino acids in the production of recombinant or synthetic formsof chimeric polypeptides according to the invention. It will be apparentto one skilled in the art that modified amino acids include, by way ofexample and not by way of limitation, 4-hydroxyproline, 5-hydroxylysine,N⁶-acetyllysine, N⁶-methyllysine, N⁶,N⁶-dimethyllysine,N⁶,N⁶,N⁶-trimethyllysine, cyclohexyalanine, D-amino acids, ornithine.

Modifications which alter the biological activity of a polypeptideaccording to the invention are also within the scope of the invention,for example, a modification which converts an agonist to an antagonist,sometimes referred to as a dominant negative mutation, or produces asuper agonist. In our co-pending application, PCT/GB02/005523, currentlyunpublished, we disclose a variant growth hormone polypeptide that actsas an antagonist. The polypeptide disclosed in PCT/GB02/005523, which isincorporated by reference, is a chimeric polypeptide comprising at leastone modified binding domain of growth hormone (GH) and a growth hormonebinding domain of a growth hormone receptor (GHR).

Modified GH's are disclosed in U.S. Pat. No. 5,849,535 which isincorporated by reference. The modification to GH is at both site 1 andsite 2 binding sites. The modifications to site 1 produce a GH moleculewhich has a higher affinity for GHR compared to wild-type GH. Thesemodified GH molecules act as agonists. There is also disclosure of site2 modifications which result in the creation of GH antagonists. Furtherexamples of modifications to GH which alter the binding affinity of GHfor site 1 are disclosed in U.S. Pat. No. 5,854,026; U.S. Pat. No.6,004,931; U.S. Pat. No. 6,022,711; U.S. Pat. No. 6,057,292; and U.S.Pat. No. 6,136,563 each of which are incorporated by reference.Modifications to site 2 are also disclosed, in particular amino acidresidue G120 in GH which when modified to either arginine, lysine,tryptophan, tyrosine, phenylalanine, or glutamic acid creates a GHmolecule with antagonistic properties. PCT/GB02/005523 discloseschimeric polypeptides which comprise modified growth hormone fused tothe extracellular binding domain of growth hormone receptor. The chimeraacts as an antagonist and has the property of reduced systemic clearancewhen administered to a patient in need of GH antagonist therapy

The current application modifies those GH variants disclosed in U.S.Pat. No. 5,849,535, U.S. Pat. No. 5,854,026; U.S. Pat. No. 6,004,931;U.S. Pat. No. 6,022,711; U.S. Pat. No. 6,057,292; and U.S. Pat. No.6,136,563 by the inclusion of a domain which comprises a heterologoussignal sequence which directs the attachment ofglycosylphosphatidylinositol. The current application also modifies thechimeric GH antagonist disclosed in PCT/GB02/005523 by the inclusion ofsaid signal sequence for the attachment of glycosylphosphatidylinositol.

In a yet further preferred embodiment of the invention there is provideda polypeptide wherein said polypeptide comprises at least twopolypeptides according to the invention which two polypeptides arelinked via a linking molecule. Preferably said linking molecule is aflexible linker. Alternatively said linking molecule is a rigid linker.

Preferably the linker comprises at least one copy of the peptide:

Gly Gly Gly Gly Ser (hereinafter referred to as “Gly4Ser”).

In a further preferred embodiment of the invention said linker comprisesat least 2, 3, 4 or 5 copies of said Gly4Ser linker.

In a yet further preferred embodiment of the invention said linkerfurther comprises a protease sensitive site. Preferably said cleavagesite is sensitive to a serum protease

Preferably said cleavage site comprises the amino acid sequence: LVPRGS,or variant thereof.

In a further preferred embodiment of the invention said cleavage sitecomprises at least one copy of the amino acid sequence: SGGGG, orfunctional variant thereof. Preferably, said cleavage site comprises theamino acid sequence PGISGGGGGG.

More preferably still said cleavage site comprises the amino acidsequence: LVPRGS PGISGGGGGG, or variant thereof.

Alternatively, said cleavage site comprises at least two copies of theamino acid sequence SGGGG, or functional variant thereof, which flanksaid cleavage site.

In a further preferred embodiment of the invention said cleavage site issensitive to the serum protease thrombin.

In a yet further preferred embodiment of the invention there is providedan oligomeric polypeptide molecule comprising a plurality ofpolypeptides according to the invention.

In a preferred embodiment said protein comprises at least 3, 4, 5, 6, 7,8, 9, or 10 polypeptides according to the invention.

According to a further aspect of the invention there is provided anucleic acid molecule comprising a nucleic acid sequence which encodes achimeric polypeptide according to the invention.

According to a yet further aspect of the invention there is provided avector comprising a nucleic acid molecule according to the invention.

In a preferred embodiment of the invention said vector is an expressionvector adapted for eukaryotic gene expression.

Typically said adaptation includes, the provision of transcriptioncontrol sequences (promoter/enhancer sequences) which mediatecell/tissue specific expression. These promoter sequences may becell/tissue specific, inducible or constitutive.

Promoter is an art recognised term and, for the sake of clarity,includes the following features which are provided by example only, andnot by way of limitation. Enhancer elements are cis acting nucleic acidsequences often found 5′ to the transcription initiation site of a gene(enhancers can also be found 3′ to a gene sequence or even located inintronic sequences and are therefore position independent). Enhancersfunction to increase the rate of transcription of the gene to which theenhancer is linked. Enhancer activity is responsive to trans actingtranscription factors (polypeptides) which have been shown to bindspecifically to enhancer elements. The binding/activity of transcriptionfactors (please see Eukaryotic Transcription Factors, by David SLatchman, Academic Press Ltd, San Diego) is responsive to a number ofenvironmental cues which include, by example and not by way oflimitation, intermediary metabolites (e.g. glucose, lipids),environmental effectors (e.g. heat).

Promoter elements also include so called TATA box and RNA polymeraseinitiation selection (RIS) sequences which function to select a site oftranscription initiation. These sequences also bind polypeptides whichfunction, inter alia, to facilitate transcription initiation selectionby RNA polymerase.

Adaptations also include the provision of selectable markers andautonomous replication sequences which both facilitate the maintenanceof said vector in either the eukaryotic cell. Vectors which aremaintained autonomously are referred to as episomal vectors.

Adaptations which facilitate the expression of vector encoded genesinclude the provision of transcription termination/polyadenylationsequences. This also includes the provision of internal ribosome entrysites (IRES) which function to maximise expression of vector encodedgenes arranged in bicistronic or multi-cistronic expression cassettes.

These adaptations are well known in the art. There is a significantamount of published literature with respect to expression vectorconstruction and recombinant DNA techniques in general. Please see,Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory, Cold Spring Harbour, N.Y. and referencestherein; Marston, F (1987) DNA Cloning Techniques: A Practical ApproachVol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

It will be apparent to one skilled in the art that the vectors accordingto the invention could be gene therapy vectors. Gene therapy vectors aretypically virus based. A number of viruses are commonly used as vectorsfor the delivery of exogenous genes. Commonly employed vectors includerecombinantly modified enveloped or non-enveloped DNA and RNA viruses,preferably selected from baculoviridiae, parvoviridiae, picornoviridiae,herpesveridiae, poxyiridae, adenoviridiae, or picornnaviridiae. Chimericvectors may also be employed which exploit advantageous elements of eachof the parent vector properties (See e.g., Feng, et al. (1997) NatureBiotechnology 15:866-870). Such viral vectors may be wild-type or may bemodified by recombinant DNA techniques to be replication deficient,conditionally replicating or replication competent.

Preferred vectors are derived from the adenoviral, adeno-associatedviral and retroviral genomes. In the most preferred practice of theinvention, the vectors are derived from the human adenovirus genome.Particularly preferred vectors are derived from the human adenovirusserotypes 2 or 5. The replicative capacity of such vectors may beattenuated (to the point of being considered “replication deficient”) bymodifications or deletions in the E1a and/or E1b coding regions. Othermodifications to the viral genome to achieve particular expressioncharacteristics or permit repeat administration or lower immune responseare preferred.

Alternatively, the viral vectors may be conditionally replicating orreplication competent. Conditionally replicating viral vectors are usedto achieve selective expression in particular cell types while avoidinguntoward broad spectrum infection. Examples of conditionally replicatingvectors are described in Pennisi, E. (1996) Science 274:342-343;Russell, and S. J. (1994) Eur. J. of Cancer 30A(8): 1165-1171.Additional examples of selectively replicating vectors include thosevectors wherein a gene essential for replication of the virus is undercontrol of a promoter which is active only in a particular cell type orcell state such that in the absence of expression of such gene, thevirus will not replicate. Examples of such vectors are described inHenderson, et al., U.S. Pat. No. 5,698,443 issued Dec. 16, 1997 andHenderson, et al., U.S. Pat. No. 5,871,726 issued Feb. 16, 1999 theentire teachings of which are herein incorporated by reference.

Additionally, the viral genome may be modified to include induciblepromoters which achieve replication or expression only under certainconditions. Examples of inducible promoters are known in the scientificliterature (See, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res.Comm. 230: 426-430; Iida, et al. (1996) J. Virol. 70(9): 6054-6059;Hwang, et al. (1997) J. Virol 71(9): 7128-7131; Lee, et al. (1997) Mol.Cell. Biol. 17(9): 5097-5105; and Dreher, et al. (1997) J. Biol. Chem272(46): 29364-29371.

Vectors may also be non-viral and are available from a number ofcommercial sources readily available to a person—skilled in the art. Forexample, the vectors may be plasmids which can be episomal orintegrating.

According to a further aspect of the invention there is provided a celltransfected with the vector or nucleic acid molecule according to theinvention.

In a preferred embodiment of the invention said cell is a eukaryoticcell.

Preferably said eukaryotic cell is selected from the group consistingof: a fungal cell eg Saccharomyces cerevisiae, Pichia spp; slime mold(eg Dictyostelium spp); insect cell (eg Spodoptera frugiperda); a plantcell; or a mammalian cell (e.g. CHO cell).

Methods to transfect cells, in particular eukaryotic cells, are wellknown in the art.

Transfection methods to introduce DNA into cells typically involve theuse of chemical reagents, cationic lipids or physical methods. Chemicalmethods which facilitate the uptake of DNA by cells include the use ofDEAE—Dextran (Vaheri and Pagano (1965) Science 175: p 434). DEAE-dextranis a negatively charged cation which associates and introduces the DNAinto cells but which can result in loss of cell viability. Calciumphosphate is also a commonly used chemical agent which whenco-precipitated with DNA introduces the DNA into cells (Graham et alVirology (1973) 52: p 456).

The use of cationic lipids (e.g. liposomes (Felgner (1987) Proc. Natl.Acad. Sci USA, 84:p 7413)) has become a common method since it does nothave the degree of toxicity shown by the above described chemicalmethods. The cationic head of the lipid associates with the negativelycharged nucleic acid backbone of the DNA to be introduced. The lipid/DNAcomplex associates with the cell membrane and fuses with the cell tointroduce the associated DNA into the cell. Liposome mediated DNAtransfer has several advantages over existing methods. For example,cells which are recalcitrant to traditional chemical methods are moreeasily transfected using liposome mediated transfer.

More recently still, physical methods to introduce DNA have becomeeffective means to reproducibly transfect cells. Direct microinjectionis one such method which can deliver DNA directly to the nucleus of acell (Capecchi (1980) Cell, 22:p 479). This allows the analysis ofsingle cell transfectants. So called “biolistic” methods physicallyshoot DNA into cells and/or organelles using a particle gun (Neumann(1982) EMBO J, 1: p 841). Electroporation is arguably the most popularmethod to transfect DNA. The method involves the use of a high voltageelectrical charge to momentarily permeabilise cell membranes making thempermeable to macromolecular complexes.

More recently still, a method termed immunoporation has become arecognised technique for the introduction of nucleic acid into cells,(see Bildirici et al Nature (2000) 405, 298). The technique involves theuse of beads coated with an antibody to a specific receptor. Thetransfection mixture includes nucleic acid, typically vector DNA,antibody coated beads and cells expressing a specific cell surfacereceptor. The coated beads bind the cell surface receptor and when ashear force is applied to the cells the beads are stripped from the cellsurface. During bead removal a transient hole is created through whichnucleic acid and/or other biological molecules can enter. Transfectionefficiency of between 40-50% is achievable depending on the nucleic acidused.

In a further aspect of the invention there is provided a method toprepare a polypeptide according to the invention comprising:

-   (i) growing a cell transfected with a vector or nucleic acid    according to the invention in conditions conducive to the    manufacture of said polypeptide; and-   (ii) purifying said polypeptide from said cell, or its growth    environment.

It will be apparent that polypeptides according to the invention can bepurified in a number of ways from cells expressing nucleic acids and/orvectors according to the invention. For example, cells maybe isolatedfrom cell growth media followed by proteolytic cleavage of saidpolypeptide from the cell membrane. Alternatively, polypeptides can besecreted or cleaved in cell culture to release the polypeptide into thesurrounding cell growth media. The polypeptides are then subsequentlyisolated from cell growth media and purified by conventional techniques(e.g. affinity chromatography, ultra centrifugation). Polypeptides canbe further processed to remove the glycosylphosphatidylinositol anchor.

According to a yet further aspect of the invention there is provided acell wherein said cell presents, at least at its cell surface, apolypeptide according to the invention.

It will be apparent to one skilled in the art that cells could beincubated with chimeric polypeptides as herein disclosed which wouldinsert via the glycosylphosphatidylinositol anchor and become localisedin the cell membrane. These cells could then act as delivery vehiclesfor said polypeptides when administered to patients in need oftreatment. For example, polypeptides according to the invention could beincubated will red blood cells taken from a patient, which issubsequently re-administered to the patient.

Also polypeptides according to the invention may insert into a cellmembrane after administration (i.e. if injected, into a joint orsystemically the GPI containing polypeptide may insert into cellmembranes. GPI containing cytokine antagonists may be injected into acoronary artery to block a local cytokine effect. A common problem nowis restonosis of coronary arteries after they have been dilated blockingthe inflammatory response by local injection of a cytokine antagonist isdesirable. The insertion could either by through admistering peptideitself or getting local expression by applying a DNA encoding for thepeptide-GPI.

In a yet further aspect of the invention there is provided apolypeptide, a nucleic acid molecule, a vector or a cell according tothe invention for use as a pharmaceutical.

Preferably said polypeptide, nucleic acid molecule, vector or cell isused in a pharmaceutical composition.

When administered the pharmaceuticals/compositions of the presentinvention is administered in pharmaceutically acceptable preparations.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, and optionally other therapeutic agents.

The pharmaceuticals/compositions of the invention can be administered byany conventional route, including injection. The administration andapplication may, for example, be oral, intravenous, intraperitoneal,intramuscular, intracavity, intraarticuar, subcutaneous, topical (eyes),dermal (e.g a cream lipid soluble insert into skin or mucus membrane) ortransdermal.

Pharmaceuticals/compositions of the invention are administered ineffective amounts. An “effective amount” is that amount of apharmaceuticals/compositions that alone, or together with further dosesor synergistic drugs, produces the desired response. This may involveonly slowing the progression of the disease temporarily, although morepreferably, it involves halting the progression of the diseasepermanently. This can be monitored by routine methods or can bemonitored according to diagnostic methods.

The doses of the pharmaceuticals/compositions administered to a subjectcan be chosen in accordance with different parameters, in particular inaccordance with the mode of administration used and the state of thesubject (i.e. age, sex). When administered, thepharmaceuticals/compositions of the invention are applied inpharmaceutically-acceptable amounts and in pharmaceutically-acceptablecompositions. Such preparations may routinely contain salts, bufferingagents, preservatives, compatible carriers, and optionally othertherapeutic agents. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically-acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically-acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic, and the like. Also,pharmaceutically-acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts.

The pharmaceuticals/compositions may be combined, if desired, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.

The pharmaceuticals/compositions may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; and phosphoric acid in a salt.

The pharmaceuticals/compositions also may contain, optionally, suitablepreservatives, such as: benzalkonium chloride; chlorobutanol; parabensand thimerosal.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous or non-aqueous preparation which ispreferably isotonic with the blood of the recipient. This preparationmay be formulated according to known methods using suitable dispersingor wetting agents and suspending agents. The sterile injectablepreparation also may be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,3-butane diol. Among the acceptable solvents that may beemployed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono-or di-glycerides. In addition,fatty acids such as oleic acid may be used in the preparation ofinjectables. Carrier formulation suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

Advantageously, pharmaceutical preparations comprising polypeptidesaccording to the invention are able to form micelles due to the presenceof glycosylphosphatidylinositol anchors. The formation of micelles,either in vitro prior to administration, or in vivo, afteradministration, enables the formation of large complexes which havereduced clearance rates. This allows the use of lower effective doses ofpolypeptide to be administered thereby reducing harmful side-effects.

An analogous effect is shown if the polypeptides according to theinvention are incorporated into liposomes. Liposomes are lipid basedvesicles which encapsulate a selected therapeutic agent which is thenintroduced into a patient. The liposome is manufactured either from purephospholipid or a mixture of phospholipid and phosphoglyceride.Typically liposomes can be manufactured with diameters of less than 200nm, this enables them to be intravenously injected and able to passthrough the pulmonary capillary bed. Furthermore the biochemical natureof liposomes confers permeability across blood vessel membranes to gainaccess to selected tissues. Liposomes do have a relatively shorthalf-life. So called STEALTH® liposomes have been developed whichcomprise liposomes coated in polyethylene glycol (PEG). The PEG treatedliposomes have a significantly increased half-life when administeredintravenously to a patient. In addition, STEALTH® liposomes show reduceduptake in the reticuloendothelial system and enhanced accumulationselected tissues. In addition, so called immuno-liposomes have beendevelop which combine lipid based vesicles with an antibody orantibodies, to increase the specificity of the delivery of the agent toa selected cell/tissue.

The use of liposomes as delivery means is described in U.S. Pat. No.5,580,575 and U.S. Pat. No. 5,542,935.

According to a yet further aspect of the invention there is provided amethod of treatment of an animal, preferably a human, comprisingadministering an effective amount of a nucleic acid and/or vector and/orpolypeptide and/or cell according to the invention.

An embodiment of the invention will now be described by example only andwith reference to the following figures;

FIG. 1 illustrates the cloning strategy to generate GPI linked proteins.

The gene of interest is positioned between a NdeI (“sticky” cutter) andEcoRV/XmaI (blunt cutter) restriction sites by PCR. The PCR product isthen digested with NdeI and EcoRV/XmaI, the resulting product is ligatedbetween NdeI and EcoRV sites in pCR-3/GHRss-GPI to obtain a vector fromwhich a GPI linked protein maybe expressed;

FIG. 2 illustrates the nucleotide and amino acid sequence of the GH-GPIconstruct.

Sequence from the original pCR-3/GPI vector is shown underlined, linkersequence between the promoter and the initiation codon, ATG, is shown inwhite on black and the subsequent GHR signal sequence is in similarcolours but italicised. The GH sequence is shown in CAPITALS and thelink between the GH protein and the GPI anchor shown in black on grey,the GPI anchor signal sequence is shown italicised and underlined. Allthe relevant restrictions sites are in bold and include BamHI (ggatcc),NdeI (catatg) and EcoRV (gatatc);

FIG. 3 illustrates the nucleotide and amino acid sequence of the 1B1-GPIconstruct (1B1 is GH linked to GHR).

Sequence from the original pCR-3/GPI vector is shown underlined, linkersequence between the promoter and the initiation codon, ATG, is shown inwhite on black and the subsequent GHR signal sequence is shown insimilar colours but italicised. The 1B1 sequence is shown in CAPITALSand the link between the 1B1 protein and the GPI anchor shown in blackon grey, the GPI anchor signal sequence is shown italicised andunderlined. All the relevant restrictions sites are in bold and includeBamHI (ggatcc), NdeI (catatg) and XmaI (cccggg);

FIG. 4 illustrates the nucleotide and amino acid sequence of the 1C1-GPIconstruct (1C1 is GH linked to GH as a tandem).

Sequence from the original pCR-3/GPI vector is shown underlined, linkersequence between the promoter and the initiation codon, ATG, is shown inwhite on black and the subsequent GHR signal sequence is shown insimilar colours but italicised. The 1C1 sequence is shown in CAPITALSand the link between the 1C1 protein and the GPI anchor shown in blackon grey, the GPI anchor signal sequence is shown italicised andunderlined. All the relevant restrictions sites are in bold and includeBamHI (ggatcc), NdeI (catatg) and EcoRV (gatatc);

FIG. 5 illustrates a charcoal assay for GHBP. The assay shows acomparison of the amounts of GHBP released into the medium fromtransient and stable transfected CHO cells after 48 hours. In all casesthe medium was concentrated 20 times using a Centricon 30 column priorto the charcoal assay being carried out; and

FIG. 6 illustrates a western blot of samples from the membranepurification of GHBP-GPI expressed in CHO cells. The western blot wasgenerating by probing with mouse anti-GHBP antibodies (2C8+263) and thenprobing with peroxidase labelled anti-mouse IgG. The lanes on the blotcontain—1. Positive control: GHBP-GPI (stable clone), 2. Untransfectedcells (P1), 3. Untransfected cells (P2), 4. Untransfected cells (S1), 5.Molecular weight standards (25, 37, 50, 75, 100, 150, 200 kDa), 6.Medium from untransfected cells, 7. GHBP-GPI (stable clone) (P1), 8.GHBP-GPI (stable clone) (P2), 9. GHBP-GPI (stable clone) (S1) and 10.Medium from GHBP-GPI (stable clone). The blot shows that GHBP-GPI hasbeen purified successfully in the membrane prep (Lane 8).

Materials and Methods

Cloning Strategy

The GHR signal sequence was first inserted into the vector, pCR-3/GPI,to enable the subsequently expressed proteins to be targeted to the cellmembrane. The GHR signal sequence flanked by a BamHI site and(NdeI-EcoRV) sites was obtained by PCR using the primers GHRss_for1 andGHRss_rev1. This insert was ligated into pCR-3/GPI between the BamHI andEcoRV sites.

The protein of interest was then ligated in-frame between the NdeI andEcoRV restriction sites between the GHR signal sequence and the Thy-1(GPI anchor signal sequence). PCR was used to generate suitablerestriction sites at either end of the gene encoding the protein ofinterest, a BamHI site was used upstream of the gene and a blunt-cuttingrestriction enzyme (EcoRV or XmaI) was used downstream of the gene beingsub-cloned (FIG. 1).

a) GH-GPI (FIG. 2)

The primers GH2GPI_for1 and GH2GPI_rev1 (Table 1) were used in a PCRreaction to amplify the hGH gene flanked by NdeI and EcoRV sites. Theresulting PCR product was digested with these restriction enzymes andthen ligated into NdeI/EcoRV double-digested pCR3-GPI. This was thenligated into E. coli XL1 Blue cells.

b) 1B1-GPI (FIG. 3). 1B1 is GH linked through its C-terminus to theextracellular domain of the GH receptor and the linked to the GPI signalsequence. Since the 1B1 gene already contains the EcoRV restrictionsite, the insert was generated between NdeI and XmaI restriction sitesusing the primers GH2GPI_for1 and 1B12GPI_rev1 (Table 1). The resultingPCR product was digested with these restriction enzymes and then ligatedinto NdeI/EcoRV double-digested pCR3-GPI. This was then ligated into E.coli XL1 Blue cells.

c) 1C1-GPI (FIG. 4). 1C1 is a tandem of GH linked through the second GHC-terminus to the GPI signal sequence. The primers GH2GPI_for1 andGH2GPI_rev1 (Table 1) were used in a PCR reaction to amplify the 1C1gene flanked by NdeI and EcoRV sites. The resulting PCR product wasdigested with these restriction enzymes and then ligated into NdeI/EcoRVdouble-digested pCR3-GPI. This was then ligated into E. coli SURE cells.

Transient Transfection into CHO Cells

CHO cells were grown to 70% confluency and then transfected with 3 μg ofvector (e.g. pCR-3/GHBP-GPI) using the LT1 Reagent Kit (CorrusScientific Ltd.). The cells were then grown overnight at 37° C.

Stable Transfection into CHO Cells

CHO cells were grown to 70% confluency and then transfected with 8 μg ofvector (e.g. pCR-3/GHBP-GPI) using the Fugene 6 methodology (Roche).After 24 hours incubation the media on the cells was replaced withselective media (CHO cell media with 400 μg/ml G418), if required thecells were split (1:3) onto fresh 100 mm dishes. The dishes wereincubated for a further 2-3 days.

The media was once again replaced with selective media, this time with1000 μg/ml G418 added, and grown for a further 2 days. The cells werethen split (1:10) onto fresh dishes with selective media and incubatedfor a further 4 days.

The media was once again replaced with selective media and the dishesincubated for a week. At this point non-transfected cells should havedied off leaving only transfected cells. The cells were then processedfor FAC sorting/analysis to determine the levels of expression.

Charcoal Assay

This assay is used to determine the amount of binding protein present ina liquid medium.

I¹²⁵ ligand (e.g. I¹²⁵ GH or I¹²⁵Leptin) was added to a solutioncontaining an unknown amount of binding protein (e.g. GHBP-GPI, Obr-GPI)and was incubated overnight at 4° C. on a rotating wheel. Dextran coatedcharcoal (Sigma, C-6197) was then added to the tube and this incubatedfor 15 minutes at room temperature on a rotating wheel. The tube wascentrifuged at 13,000 rpm for 12 minutes and the supernatant removed toa new tube. The Total Binding (TB) was then counted using agamma-counter.

The Non-Specfic Binding (NSB) was also measured by repeating the aboveprocedure but adding a large excess of ‘cold’ ligand in addition to theI¹²⁵ligand. I¹²⁵ligand alone was also processed in this way, however thedextran-coated charcoal was not added to this sample, this gave theTotal Counts (TC).

The Percentage Specific Binding (PSB) was calculated using the followingformula:—PSB=(TB−NSB/TC)×100For each sample the charcoal assay was done in triplicate and the meanPSB reported with the calculated standard error.

This assay was used to measure the amount of GHBP released in the mediumfrom cells transfected (transient and stable) with pCR-3/GHBP-GPI after48 hours, the medium was concentrated 20 times using a Centricon 30column prior to being assayed. Medium from cells transfected withpCR-3/GPI was also processed in the same way (FIG. 5).

Purification

a) Preparation of Soluble and Membranes Fractions from CHO-K1 Cell LineStably Expressing GHBP-GPI

CHO-K1 cells either non-transfected or expressing GHBP-GPI were grown toconfluency on 100 cm dishes. After serum starvation for 24-48 hrs theculture medium was removed and concentrated and desalted using aCentricon YM-10 filtration column and frozen at −80° C.

For preparation of membranes the cells were first washed with PBSfollowed by fresh PBS containing a protease inhibitor cocktail (1 μg/mlaprotinin, antipain, pepstatin, leupeptin, 156.5 μg/ml Benzamidine-HCland 40 μg/ml PMSF, this is referred to as “PBS complete”). The excessPBS was drained off and the cells scraped from the plates and lysedusing a dounce homogeniser.

The lysate was firstly subjected to a low speed spin at 2.5 k rpm for 10minutes, 4° C. The resultant pellet contains nuclear and cellular debris(P1 fraction). The supernatant (S1 fraction) was cloudy and containedcytosolic and membrane bound fractions. The S1 fraction was collectedand subjected to a high speed spin at 40 k rpm for 1 hour, 4° C. Thesupernatant (S2 fraction, containing soluble cytosolic material) wascollected and frozen at −80° C. The pellet (P2 fraction, containinginsoluble membrane bound material) was washed with PBS complete andcentrifuged again as before.

The P2 fraction was resuspended in PBS complete with the addition of0.1% (v/v) Triton X-100 and frozen in aliquots −80° C. Expression ofGHBP was confirmed by Western blotting using anti GHBP antibodies (FIG.6).

b) Preparation of GH Affinity Column

GH was covalently coupled to CNBr activated sepharose by the followingmethod: Breifly, 0.5 g of CNBr activated sepharose 4 fast flow (Sigma:C-5338, lot no: 91K1548) was resuspended in 10 ml ice cold 1 mM HCl, theswollen matrix was then washed with 100 ml of the same solution. Thewashed matrix was then immediately added, to a 3 ml solution of 0.1MNaHCO₃/0.5M NaCl, pH 8.3 (coupling buffer) containing a total of 3 mgrhGH. The solution was mixed gently for 2 hrs at room temperature andsubsequently washed with coupling buffer. Any remaining active groupswere blocked by incubation with 0.2M glycine, pH 8.0 for 2 hrs at roomtemperature. To remove non-specifically bound protein the matrix wasalternately washed with coupling buffer followed by 0.1M sodiumacetate/0.5M NaCl, pH 4.5. The matrix was stored in PBS, pH7.4,containing 0.02% NaN₃ at 4° C.

c) Purification of GHBP from CHO-K1 Media

The GH-coupled column was equilibrated with 10× column volumes of PBS at4° C. Concentrated and desalted media containing GHBP was allowed toflow through the column under gravity flow. This process was repeatedfor up to 4 times. The column was washed with PBS and bound proteineluted with 3M KSCN again under gravity flow. The resultant eluate wasdesalted and concentrated using an Amicon ultrafree-MC centrifugalfiltration unit (30,000 molecular weight cut off) and samples analysedby SDS-PAGE and western blotting techniques.

This methodology was also utilised to purify membrane bound protein,however 0.1% Triton was present in all buffers.

1. A chimeric polypeptide which is engineered to include a domaincomprising a sequence that directs the attachment of at least oneglycosylphosphatidylinositol molecule, wherein said polypeptide is not aligand binding domain of a cytokine receptor and is for use as apharmaceutical.
 2. The polypeptide according to claim 1 wherein saidpolypeptide is a cytokine or variant thereof.
 3. The polypeptideaccording to claim 1 wherein said domain comprises the amino acidsequence: (SEQ ID NO: 12) PSPTPTETAT PSPTPKPTST PEETEAPSSA TTLISPLSLIVIFISFVLLI.


4. The polypeptide according to claim 1 wherein said domain comprisesthe amino acid sequence: (SEQ ID NO: 13) LVPRGSIEGR GTSITAYNSEGESAEFFFLL ILLLLLVLV.


5. The polypeptide according to claim 1 wherein said domain comprisesthe amino acid sequence: TSITAYKSE GESAEFFFLL ILLLLLVLV. (SEQ ID NO: 14)


6. The polypeptide according to claim 1 wherein said polypeptideincludes at least one glycosylphosphatidylinositol molecule.
 7. Thepolypeptide according to claim 2 wherein said polypeptide is selectedfrom the group consisting of: growth hormone; leptin; erythropoietin;prolactin; TNF, interleukins (IL), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-9, IL-10, IL-11; the p35 subunit of IL-12, IL-13, IL-15; granulocytecolony stimulating factor (G-CSF); granulocyte macrophage colonystimulating factor (GM-CSF); ciliary neurotrophic factor (CNTF);cardiotrophin-1 (CT-1); leukemia inhibitory factor (LIF); oncostatin M(OSM); interferon, IFNα and IFNγ,
 8. The polypeptide according to claim1 wherein said polypeptide has been modified by addition, deletion orsubstitution of at least one amino acid residue to provide a sequencevariant of said polypeptide.
 9. The polypeptide according to claim 8wherein said variant polypeptide is growth hormone which has beenmodified in at least one growth hormone receptor binding domain.
 10. Thepolypeptide according to claim 9 wherein said growth hormone receptorbinding domain is in site 1 of growth hormone.
 11. The polypeptideaccording to claim 9 wherein said growth hormone receptor binding domainis modified in site 2 of growth hormone.
 12. The polypeptide accordingto claim 9 wherein said growth hormone receptor binding domain ismodified in site 1 and site 2 of growth hormone.
 13. The polypeptideaccording to claim 10 wherein said modification is selected from thegroup consisting of: histidine 18 with alanine or aspartic acid; and/orhistidine 21 with asparagine; and/or glutamine 22 with alanine; and/orphenylalanine 25 with alanine; and/or aspartic acid 26 with alanine;and/or glutamine 29 with alanine; and/or glutamic acid 167 with alanine;and/or aspartic acid 171 with serine; and/or lysine 172 with serine oralanine; and/or isoleucine 179 with tyrosine, as represented by thegrowth hormone amino acid sequence in FIG. 2 (amino acids 21-254 of SEQID NO: 2).
 14. The polypeptide according to claim 13 wherein saidmodification consists of the the amino acid substitutions: histidine 18aspartic acid; histidine 21 asparagine; arginine 167 asparagine;aspartic acid 171 arginine; glutamic acid 174 serine; and isoleucine 179threonine; as represented by the GH amino acid sequence in FIG. 2 (aminoacids 21-254 of SEQ ID NO: 2).
 15. The polypeptide according to claim 13wherein said modification consists of the amino acid substitutions:histidine 18 alanine; glutamine 22 alanine; phenylalanine 25 alanine;aspartic acid 26 alanine; glutamine 29 alanine; glutamic acid 65alanine; lysine 168 alanine; and glutamic acid 174 alanine; asrepresented by the GH amino acid sequence in FIG. 2 (amino acids 21-254of SEQ ID NO: 2).
 16. The polypeptide according to claim 11 wherein saidsite 2 modification is to amino acid residue glycine 120 of the aminoacid sequence presented in FIG. 2 (amino acids 21-254 of SEQ ID NO: 2).17. The polypeptide according to claim 16 wherein said site 2modification is a substitution of glycine for an amino acid selectedfrom the group consisting of: arginine; alanine; lysine; tryptophan;tyrosine; phenylalanine; and glutamic acid.
 18. The polypeptideaccording to claim 17 wherein said site 2 substitution is glycine 120for arginine or lysine or alanine.
 19. The polypeptide according toclaim 1 wherein said polypeptide is an antibody.
 20. The polypeptideaccording to claim 19 wherein said antibody is a monoclonal antibody, orthe active binding fragment thereof.
 21. The polypeptide according toclaim 20 wherein said monoclonal antibody is a humanised antibody. 22.The polypeptide according to claim 20 wherein said monoclonal antibodyis a chimeric antibody.
 23. The polypeptide according to claim 20wherein the active the active binding fragment is selected from thegroup consisting of: F(ab′)₂, Fab, Fv and Fd fragments; CDR3 regions;and single chain antibody fragments.
 24. The polypeptide according toclaim 23 wherein said fragment is a single chain antibody fragment. 25.An oligomeric polypeptide wherein said polypeptide comprises at leasttwo polypeptides according to claim 1 which two polypeptides are linkedvia a linking molecule.
 26. The oligomeric polypeptide according toclaim 25 wherein said linker comprises at least one copy of the peptide:Gly Gly Gly Gly Ser. (SEQ ID NO: 15)


27. The oligomeric polypeptide according to claim 26 wherein said linkercomprises at least 2, 3, 4 or 5 copies of said linker.
 28. Theoligomeric polypeptide according to claim 25 wherein said linker furthercomprises a protease sensitive cleavage site.
 29. The oligomericpolypeptide according to claim 28 wherein said cleavage site issensitive to a serum protease.
 30. The oligomeric polypeptide accordingto claim 29 wherein said cleavage site comprises the amino acidsequence: LVPRGS. (SEQ ID NO: 16)


31. The oligomeric polypeptide according to claim 29 wherein saidcleavage site comprises the amino acid sequence PGISGGGGGGSGGGG. (SEQ IDNO: 20)


32. The oligomeric polypeptide according to claim 29 wherein saidcleavage site comprises the amino acid sequence: LVPRGS PGISGGGGGG. (SEQID NO: 19)


33. The oligomeric polypeptide according to claim 29 wherein saidcleavage site comprises at least two copies of the amino acid sequenceSGGGG (SEQ ID NO: 17) which flank said cleavage site.
 34. An isolatednucleic acid molecule comprising a nucleic acid sequence which encodesthe polypeptide according to claim
 1. 35. A vector comprising thenucleic acid molecule according to claim
 34. 36. The vector according toclaim 35 wherein said vector is an expression vector adapted foreukaryotic gene expression.
 37. A cell transfected with the nucleic acidmolecule of claim
 34. 38. A method to prepare the polypeptide of claim1, comprising: i) growing a cell transfected with a nucleic acidsequence which encodes the polypeptide of claim 1 in conditionsconducive to the manufacture of said polypeptide; and ii) purifying saidpolypeptide from said cell, or its growth environment.
 39. A cellwherein said cell presents, at least at its cell surface, a polypeptideor oligomeric polypeptide according to claim
 1. 40. A method oftreatment of an animal comprising administering an effective amount ofthe isolated nucleic acid molecule of claim
 34. 41. A method oftreatment of an animal, comprising administering an effective amount ofthe polypeptide of claim
 1. 42. A method of treatment of an animal,comprising administering an effective amount of the cell of claim 39.